Wild-type (wt) p53 is a sequence-specific DNA binding protein found in humans and other mammals, which has tumor suppressor function [See, e.g., Harris (1993), Science, 262:1980-1981]. The wild-type p53 protein functions to regulate cell proliferation and cell death (also known as apoptosis). It also participates in the response of the cell to DNA damaging agents [Harris (1993), cited above]. In more than half of all human tumors p53 is inactivated by mutations and is therefore unable to arrest cell proliferation or induce apoptosis in response to DNA damaging agents, such as radiation and chemotherapeutics commonly used for cancer treatment. The nucleotide and amino acid sequences of human p53 are reported below as SEQ ID NOS: 1 and 2, respectively [Zakut-Houri et al, (1985), EMBO J., 4:1251-1255; GenBank Code Hsp53]. The amino acid sequence of p53 is conserved across evolution [Soussi et al, (1990), Oncogene, 5:945-952], suggesting that its function is also conserved.
At the biochemical level, p53 is a tetrameric DNA sequence-specific transcription factor. Its DNA binding and transcriptional activities are required for p53 to suppress tumor growth [Pietenpol et al, (1994), Proc. Natl. Acad. Sci. USA, 91:1998-2002]. p53 forms homotetramers in the absence of DNA and maintains its tetrameric stoichiometry when bound to DNA [Kraiss et al, (1988), J. Virol., 62:4737-4744; Stenger et al, (1992), Mol. Carcinog., 5:102-106; Sturzbecher et al, (1992), Oncogene, 7:1513-1523; Friedman et al, (1993), Proc. Natl. Acad. Sci. USA, 90:3319-3323; Halazonetis and Kandil (1993), EMBO J., 12:5057-5064; and Hainaut et al, (1994), Oncogene, 9:299-303]. Consistent with the observation that p53 binds DNA as a homotetramer, the known physiologically relevant DNA sites recognized by p53 contain four pentanucleotide repeats [El-DeiryDeiry et al, (1993), Cell, 75:817-825; Wu et al, (1993), Genes Dev., 7:1126-1132; Kastan et al, (1992), Cell, 71:587-597]. Each pentanucleotide repeat is recognized by one subunit of the p53 homotetramer [Halazonetis and Kandil (1993), cited above; Cho et al, (1994), Science, 265:346-355]. The ability of p53 to bind DNA in a sequence-specific manner maps to amino acid residues 90-290 of SEQ ID NO: 2 [Halazonetis and Kandil (1993), cited above; Pavletich et al, (1993), Genes Dev., 7:2556-2564; Wang et al, (1993), Genes Dev., 7:2575-2586].
Once bound to DNA, p53 activates gene transcription from neighboring promoters. The ability of p53 to activate gene transcription has been mapped to within amino acid residues 1-90 of SEQ ID NO: 2 [Fields et al, (1990), Science, 249:1046-1049].
The C-terminus of the human p53 tumor suppressor protein (i.e., amino acids 290-393 of human p53, SEQ ID NO: 2) has two functions. It induces p53 oligomerization and it regulates p53 DNA binding by controlling the conformation of p53 tetramers. These two functions map to independent regions. Oligomerization maps to amino acid residues 322-355 of SEQ ID NO: 2 [Wang et al, (1994), Mol. Cell. Biol., 14:5182-5191; Clore et al, (1994), Science, 265:386-391]. Regulation of DNA binding maps to amino acid residues 364-393 of human p53 [SEQ ID NO: 2] or to the corresponding region encompassing residues 361-390 of mouse p53 [SEQ ID NO: 15] [Hupp et al, (1992), Cell, 71:875-886; Halazonetis et al, (1993), EMBO J., 12:1021-1028; Halazonetis and Kandil (1993), cited above; Genbank locus Mmp53r].
Mutations of the p53 protein in most human tumors involve the sequence-specific DNA binding domain, so that the mutant proteins are unable to bind DNA [Bargonetti et al, (1992), Genes Dev., 6:1886-1898]. The loss of p53 function is critical for tumor development. Introduction of wild-type p53 into tumor cells leads to arrest of cell proliferation or cell death [Finlay et al, (1989), Cell, 57:1083-1093; Eliyahu et al, (1989), Proc. Natl. Acad. Sci. USA, 86:8763-8767; Baker et al, (1990), Science, 249:912-915; Mercer et al, (1990), Proc. Natl. Acad. Sci. USA, 87:6166-6170; Diller et al, (1990), Mol. Cell. Biol., 10:5772-5781; Isaacs et al, (1991), Cancer Res., 51:4716-4720; Yonish-Rouach et al, (1993), Mol. Cell. Biol., 13:1415-1423; Lowe et al, (1993), Cell, 74:957-967; Fujiwara et al, (1993), Cancer Res., 53:4129-4133; Fujiwara et al, (1994), Cancer Res., 54:2287-2291]. Thus, introduction of wild-type p53 into tumor cells has been proposed to be a viable approach to treat human cancer [see, e.g., International Patent Applications WO 9406910 A, WO 9416716 A, WO 9322430 A1, EP 390323, and EP 475623 A1].
However, most tumors express mutant versions of p53 at high levels [Harris (1993), cited above]. Because these p53 mutants have intact oligomerization domains, they form hetero-tetramers with wild-type p53. Such hetero-tetramers are biochemically inactive or characterized by considerably reduced activity compared to wild-type p53 tetramers [Milner and Medcalf (1991), Cell, 65:765-774; Bargonetti et al, (1992), cited above; Farmer et al, (1992), Nature, 358:83-86; Kern et al, (1992), Science, 256:827-830]. Thus, if one were to treat human cancer by introduction of wild-type p53 in tumor cells, the effectiveness of this therapeutic approach would be limited by the presence of mutant p53 in the cancer cells.
Thus, there is a need in the art for the identification of compositions which are not inhibited by endogenous p53, as well as for methods for the uses of such compositions for therapeutic purposes.